PROJECT 3 DESCRIPTION (provided by applicant): Francisella tularensis is a gram-negative bacterium that is the causative agent of tularemia, a highly infectious disease that can be fatal in humans, particularly in the pneumonic form. F. tularensis is an ideal bioweapon because of its extremely low LD50 and ease of aerosol dissemination. The disease is treatable with antibiotics if diagnosed early, but there is no licensed vaccine currently available and, therefore the population is at considerable risk in case of mass exposure. Many studies have focused on immunity to an attenuated live vaccine strain (LVS) derived from F. tularensis subsp. holoarctica, but virtually nothing is known about the immune response during pneumonic tularemia caused by the highly virulent F. tularensis subsp. tularensis. Toll-like receptors (TLRs) are critical innate immunity response elements that serve as a first line of defense by recognizing conserved, repeated patterns in molecules expressed by many pathogens. TLRs signal cells of the innate response and shape subsequent adaptive immunity by inducing expression of chemokines, pro-inflammatory cytokines and co-stimulatory molecules and thus are important targets for new adjuvants and vaccines. Our preliminary studies demonstrate a defect in the ability of primary alveolar macrophages from MyD88 mice to secrete TNF-a upon infection with F. tularensis LVS and increased susceptibility of MyD88-/- and TLR2-/- mice to LVS infection. Recent published studies suggest that F. tularensis may evade host innate immune responses. The central hypothesis for the proposed work is that activation/inhibition of TLR signaling plays a critical role in determining the nature of the innate response in the lung to aerosol infection by virulent F. tularensis. The proposed studies will test this hypothesis in an aerosol infection model of pneumonic tularemia in the mouse. We will 1) determine the role of TLR signaling in the host response to pulmonary infection with F. tularensis subsp. tularensis (Schu4) using KO mice and SiRNA; 2) identify and characterize TLR-regulated immune effector mechanisms that are involved in the innate response to pulmonary tularemia; and 3) define TLR signal-modulating mechanisms by which F. tularensis evades the host innate immune response. We expect that these studies will provide significant advances in our understanding of the overall immunopathogenesis of virulent F. tularensis and will provide critical insight into the role of Toll-like receptors in the host innate response to pneumonic tularemia.